Probes for INS

During collective cell migration individual cells display diverse behaviors that complicate our understanding of group cell decisions of direction and cohesion. In vivo gene and protein expression analyses would shed light on the underlying molecular choreography. However, this information has been limited due to difficulties to integrate single cell detection methods and the simultaneous readout of signals deep within the embryo. Here, we optimize and integrate multiplex fluorescence in situ hybridization by RNAscope, immunohistochemistry, and tissue clearing to visualize transcript and protein localization within single cells deep within intact chick embryos. Using standard confocal microscopy, we visualize the mRNA expression of up to 3 genes simultaneously within protein labeled HNK1-positive migrating cranial neural crest cells within 2day old cleared chick embryos. Gene expression differences measured between adjacent cells or within subregions are quantified using spot counting and polyline kymograph methods, respectively. This optimization and integration of methods provide an improved 3D in vivo molecular interrogation of collective cell migration and foundation to broaden into a wider range of embryo and adult model systems.

Alveoli, the lung's respiratory units, are tiny sacs where oxygen enters the bloodstream. They are lined by flat AT1 cells, which mediate gas exchange, and AT2 cells, which secret surfactant. Rare AT2s also function as alveolar stem cells. We show that AT2 lung stem cells display active Wnt signaling and many of them are near single, Wnt-expressing fibroblasts. Blocking Wnt secretion depletes these stem cells. Daughter cells leaving the Wnt niche transdifferentiate into AT1s: maintaining Wnt signaling prevents transdifferentiation whereas abrogating Wnt signaling promotes it. Injury induces AT2 autocrine Wnts, recruiting 'bulk' AT2s as progenitors. Thus, individual AT2 stem cells reside in single cell fibroblast niches providing juxtacrine Wnts that maintain them, whereas injury induces autocrine Wnts that transiently expand the progenitor pool. This simple niche maintains the gas exchange surface, and is coopted in cancer.